Global analysis of the genetic and epigenetic contributions to phenotypic plasticity in a wild yeast strain

The natural M28 Saccharomyces cerevisiae strain, isolated from damaged grapes in Montalcino area (Tuscany), showed a fascinating 2:2 segregation of two unlinked loci: the recessive resistance to a toxic analogue of leucine (5’5’5 trifloroleucine, TFL) and the morphotype. Two of the four M28 meiotic products (spores) in fact grow as smooth colonies while the other two exhibit complex structured filigreed colonies. In this work, preliminary phenotypic investigations demonstrated the uniqueness of the M28 system since the four meiotic products displayed a remarkable phenotypic variability concerning the ability to generate pseudohyphal structures or to invade solid substrates. Additionally, we showed that each spore is able to spontaneously and reversibly switch between the two described morphotypes at high frequencies. Seduced by this puzzling system where Mendelian and Lamarckian theories seems to be reconciled we started a global investigation of the genetic, proteomic and chromatin profiles across the four spores. Using cutting-edge mass spectrometry and sequencing technologies we obtained the following results: i) we confirmed the genetic determinant of the TFL-phenotype additionally illustrating how this loss of function can massively reshape the amino acid metabolism through the alteration of chromatin accessibility via histone modifications (trimethylation of lysine 4 of histone H3 and acetylation of histone H4); ii) we demonstrated there are no genetic polymorphisms segregating with the morphotype but rather we identified few epialleles segregating 2:2 with the morphotype; iii) for the first time we showed the extensive epigenome variability among four meiotic products of a natural S. cerevisiae strain; iv) we found structural protein rearrangements in several prion-like proteins during the dimorphic transition from filigreed to smooth and vice-versa and showed that for Hrp1 protein this switch is accompanied by a significant change in the number of cells per population bearing the Hrp1p-aggregate. These results evidenced that both genetic and epigenetic contributions are affecting the phenotypic plasticity in the M28 system proposing an update of the “genome renewal” theory that introduces chromatin reshaping as a rapid adaptive mechanism allowing the appearance of new stable and potentially advantageous traits in the progeny of diploid strains

Role of high dose octreotide LAR for the treatment of GEP-NETs

Neuroendocrine Tumours (NETs) are a heterogeneous group of rare neoplasms that account for 0,5% of all malignancies. The increased incidence observed in the last few decades may be accounted for by increased awareness, improved diagnostic tools and a revision in the definition. The main primary sites are the gastro-entero-pancreatic (GEP) tract (62-67%), and the lung (22-27%). In patients with GEP-NETs, the strongest predictor of 5-years survival is the staging. An adequate clinical management of GEP-NETs should be multidisciplinary and should aim at assuring a good quality of life. Somatostatin (sst) analogues are widely used in these tumours, which often express sst receptors, since they are demonstrated to reduce clinical symptoms and tumour growth. Herein we explore the usefulness of doubling octreotide LAR dose in selected patients after escaping from symptoms control and/or tumour stabilization in course of treatment with standard dose

Cell-Cycle Inhibition by Helicobacter pylori L-Asparaginase

Helicobacter pylori (H. pylori) is a major human pathogen causing chronic gastritis, peptic ulcer, gastric cancer, and mucosa-associated lymphoid tissue lymphoma. One of the mechanisms whereby it induces damage depends on its interference with proliferation of host tissues. We here describe the discovery of a novel bacterial factor able to inhibit the cell-cycle of exposed cells, both of gastric and non-gastric origin. An integrated approach was adopted to isolate and characterise the molecule from the bacterial culture filtrate produced in a protein-free medium: size-exclusion chromatography, non-reducing gel electrophoresis, mass spectrometry, mutant analysis, recombinant protein expression and enzymatic assays. L-asparaginase was identified as the factor responsible for cell-cycle inhibition of fibroblasts and gastric cell lines. Its effect on cell-cycle was confirmed by inhibitors, a knockout strain and the action of recombinant L-asparaginase on cell lines. Interference with cell-cycle in vitro depended on cell genotype and was related to the expression levels of the concurrent enzyme asparagine synthetase. Bacterial subcellular distribution of L-asparaginase was also analysed along with its immunogenicity. H. pylori L-asparaginase is a novel antigen that functions as a cell-cycle inhibitor of fibroblasts and gastric cell lines. We give evidence supporting a role in the pathogenesis of H. pylori-related diseases and discuss its potential diagnostic application

OPA1 Isoforms in the Hierarchical Organization of Mitochondrial Functions

OPA1 is a GTPase that controls mitochondrial fusion, cristae integrity, and mtDNA maintenance. In humans, eight isoforms are expressed as combinations of long and short forms, but it is unclear whether OPA1 functions are associated with specific isoforms and/or domains. To address this, we expressed each of the eight isoforms or different constructs of isoform 1 in Opa1−/− MEFs. We observed that any isoform could restore cristae structure, mtDNA abundance, and energetic efficiency independently of mitochondrial network morphology. Long forms supported mitochondrial fusion; short forms were better able to restore energetic efficiency. The complete rescue of mitochondrial network morphology required a balance of long and short forms of at least two isoforms, as shown by combinatorial isoform silencing and co-expression experiments. Thus, multiple OPA1 isoforms are required for mitochondrial dynamics, while any single isoform can support all other functions. These findings will be useful in designing gene therapies for patients with OPA1 haploinsufficiency

Mesenchymal Stromal Cells Primed with Paclitaxel Provide a New Approach for Cancer Therapy

BACKGROUND: Mesenchymal stromal cells may represent an ideal candidate to deliver anti-cancer drugs. In a previous study, we demonstrated that exposure of mouse bone marrow derived stromal cells to Doxorubicin led them to acquire anti-proliferative potential towards co-cultured haematopoietic stem cells (HSCs). We thus hypothesized whether freshly isolated human bone marrow Mesenchymal stem cells (hMSCs) and mature murine stromal cells (SR4987 line) primed in vitro with anti-cancer drugs and then localized near cancer cells, could inhibit proliferation. METHODS AND PRINCIPAL FINDINGS: Paclitaxel (PTX) was used to prime culture of hMSCs and SR4987. Incorporation of PTX into hMSCs was studied by using FICT-labelled-PTX and analyzed by FACS and confocal microscopy. Release of PTX in culture medium by PTX primed hMSCs (hMSCsPTX) was investigated by HPLC. Culture of Endothelial cells (ECs) and aorta ring assay were used to test the anti-angiogenic activity of hMSCsPTX and PTX primed SR4987(SR4987PTX), while anti-tumor activity was tested in vitro on the proliferation of different tumor cell lines and in vivo by co-transplanting hMSCsPTX and SR4987PTX with cancer cells in mice. Nevertheless, despite a loss of cells due to chemo-induced apoptosis, both hMSCs and SR4987 were able to rapidly incorporate PTX and could slowly release PTX in the culture medium in a time dependent manner. PTX primed cells acquired a potent anti-tumor and anti-angiogenic activity in vitro that was dose dependent, and demonstrable by using their conditioned medium or by co-culture assay. Finally, hMSCsPTX and SR4987PTX co-injected with human cancer cells (DU145 and U87MG) and mouse melanoma cells (B16) in immunodeficient and in syngenic mice significantly delayed tumor takes and reduced tumor growth. CONCLUSIONS: These data demonstrate, for the first time, that without any genetic manipulation, mesenchymal stromal cells can uptake and subsequently slowly release PTX. This may lead to potential new tools to increase efficacy of cancer therapy

Global analysis of the genetic and epigenetic contributions to phenotypic plasticity in a wild yeast strain

The natural M28 Saccharomyces cerevisiae strain, isolated from damaged grapes in Montalcino area (Tuscany), showed a fascinating 2:2 segregation of two unlinked loci: the recessive resistance to a toxic analogue of leucine (5’5’5 trifloroleucine, TFL) and the morphotype. Two of the four M28 meiotic products (spores) in fact grow as smooth colonies while the other two exhibit complex structured filigreed colonies. In this work, preliminary phenotypic investigations demonstrated the uniqueness of the M28 system since the four meiotic products displayed a remarkable phenotypic variability concerning the ability to generate pseudohyphal structures or to invade solid substrates. Additionally, we showed that each spore is able to spontaneously and reversibly switch between the two described morphotypes at high frequencies. Seduced by this puzzling system where Mendelian and Lamarckian theories seems to be reconciled we started a global investigation of the genetic, proteomic and chromatin profiles across the four spores. Using cutting-edge mass spectrometry and sequencing technologies we obtained the following results: i) we confirmed the genetic determinant of the TFL-phenotype additionally illustrating how this loss of function can massively reshape the amino acid metabolism through the alteration of chromatin accessibility via histone modifications (trimethylation of lysine 4 of histone H3 and acetylation of histone H4); ii) we demonstrated there are no genetic polymorphisms segregating with the morphotype but rather we identified few epialleles segregating 2:2 with the morphotype; iii) for the first time we showed the extensive epigenome variability among four meiotic products of a natural S. cerevisiae strain; iv) we found structural protein rearrangements in several prion-like proteins during the dimorphic transition from filigreed to smooth and vice-versa and showed that for Hrp1 protein this switch is accompanied by a significant change in the number of cells per population bearing the Hrp1p-aggregate. These results evidenced that both genetic and epigenetic contributions are affecting the phenotypic plasticity in the M28 system proposing an update of the “genome renewal” theory that introduces chromatin reshaping as a rapid adaptive mechanism allowing the appearance of new stable and potentially advantageous traits in the progeny of diploid strains.M28 è un ceppo naturale di Saccharomyces cerevisiae, isolato da vitigni di Montalcino (Toscana), che mostra una affascinante segregazione 2:2 di due tratti non collegati tra loro: la resistenza recessiva ad un analogo tossico della leucina (5’5’5 trifloroleucina, TFL) ed il morfotipo di colonia. Due dei quattro prodotti meiotici (spore) di M28, infatti, crescono generando colonie lisce mentre gli altri due generano complesse strutture filamentose. Analisi preliminari a questo lavoro di tesi hanno dimostrato l’unicità di questo sistema data dall’osservazione che i quattro prodotti meiotici di M28 mostrano una elevata variabilità fenotipica riguardo l’abilità di differenziare in strutture pseudo-ifali nonché la capacità di invadere substrati solidi. Abbiamo successivamente dimostrato che ogni spora è capace di passare da un fenotipo di colonia liscio ad un rugoso, e viceversa, in un processo spontaneo e reversibile che avviene ad alte frequenze. Sedotti da questo intrigante sistema dove le teorie Lamarckiane e Darwiniane sembrano riconciliarsi abbiamo iniziato una investigazione globale volta ad investigare il genoma, il proteoma e la struttura cromatinica di tutti e quattro i prodotti meiotici di una tetrade di M28. Abbiamo così utilizzato tecnologie all’avanguardia sia nel campo della spettrometria di massa che del sequenziamento di ultima generazione, ottenendo i seguenti risultati: i) abbiamo confermato il determinante genetico della resistenza alla TFL mostrando inoltre come una mutazione associata alla perdita di funzione di un gene possa interferire sul metabolismo cellulare, degli amino acidi in questo caso, alterando l’accessibilità della cromatina attraverso modifiche istoniche quali la bi- e tri-metilazione della lisina 4 dell’istone H3 o l’acetilazione dell’istone H4; ii) abbiamo dimostrato che non vi sono polimorfismi che segregano con il tratto “morfologia di colonia” ma piuttosto abbiamo identificato alcuni epi-alleli che segregrano 2:2 con il morfotipo e che potrebbero essere quindi responsabili della segregazione osservata; iii) abbiamo mostrato per la prima volta l’elevata variabilità a livello dell’epigenoma di quattro prodotti meiotici di un ceppo naturale di S. cerevisiae; iv) abbiamo identificato riarrangiamenti proteici strutturali, a carico di alcune proteine associabili ai prioni, durante la transizione da morfotipo di colonia filamentoso a liscio (e viceversa) mostrando che la proteina Hrp1 genera aggregati proteici a localizzazione nucleare il cui numero cambia statisticamente durante la transizione dimorfica. In conclusione i risultati di questo lavoro evidenziano come meccanismi di tipo genetico ed epigenetico possano regolare la plasticità fenotipica in M28 portandoci ad ipotizzare un aggiornamento della teoria del “rinnovamento del genoma” che introduca le variazioni della struttura cromatinica come un meccanismo di adattamento evolutivo che permette l’insorgere di nuovi tratti, potenzialmente vantaggiosi, nella progenie di ceppi di lievito diplodi